Homeobox genes, Hox genes and apoptosis

The homeobox and the homeodomain

Development depends on controlled gene expression as well as cell division. Homeobox genes code for transcription factors that regulate groups of other genes, allowing a single regulatory gene to influence the identity and development of a body region.

The homeobox is a sequence of 180 base pairs. It lies within a gene rather than constituting the whole of one: the genes that carry it are thousands of bases long. Three bases code for one amino acid, so 180 bases code for 60, and those 60 amino acids in the finished protein are the homeodomain.

The homeodomain folds into a compact shape, three short helices with a turn between two of them, and one of those helices sits into the major groove of a DNA double helix. A protein that binds to DNA at a particular sequence and alters whether the gene there is transcribed is a transcription factor, and that is what a homeobox gene codes for. A homeobox gene therefore contributes no structural component itself; it regulates the transcription of other genes, whose products build the structure.

The sequence is also extraordinarily conserved. The homeobox of a mouse gene and the homeobox of the fruit fly gene that answers to it differ by only a handful of bases, across animals whose ancestors parted company well over five hundred million years ago, and the mouse version can be put into a fly embryo and do the fly gene's job. Conservation on that scale is evidence of two things at once: that the animals share a common ancestor which already had these genes, and that almost any change to them is fatal, so almost none of the changes that arose was ever passed on.

Homeobox

A sequence of 180 base pairs found within many genes that control development, coding for the part of the protein that binds to DNA.

Homeodomain

The 60 amino acid section of a protein coded for by a homeobox, which folds into a shape that binds to a specific DNA sequence.

Transcription factor

A protein that binds to DNA near a gene and controls whether that gene is transcribed into mRNA.

Conserved sequence

A sequence of bases that has changed very little between species over long periods, because nearly every mutation in it is harmful and is not passed on.

Hox genes and colinearity

Homeobox genes form a large family, and Hox genes are the group that helps specify what each region along the anterior-posterior axis of an animal becomes. They are not scattered through the genome; they lie together in a cluster, and the order within that cluster is examinable.

This match between the two orders is called colinearity. The gene at one end of the cluster is expressed in and specifies the most anterior region, the next gene along specifies the next region back, and the gene at the far end specifies the tail end. Nothing about a gene's chemistry requires it to sit where it does, so the arrangement is a piece of history: the cluster arose by one ancestral gene being duplicated along a chromosome, and each copy took on the region next along.

The fruit fly has one such cluster, in two pieces. A mouse and a human have four clusters, on four different chromosomes, holding thirty-nine Hox genes between them, which is the same cluster duplicated whole twice over in an early vertebrate. Each of the four keeps its internal order, so colinearity survived the duplications.

A Hox gene acts as a transcription factor within its region. Its protein binds to the control sequences of many other genes and increases their transcription, and the products of those genes determine that the region develops as a thorax rather than a head: the cells there divide on a particular schedule, differentiate into particular types and form particular structures. A mutation in one such gene can therefore have a large effect, because it alters the expression of every gene downstream of it.

The clearest evidence comes from mutations that give a region the identity of another region. A fly carrying a particular mutation in the gene called Antennapedia grows a pair of legs on its head where its antennae should be. The legs are complete and normally formed, so the genes that build a leg are intact; they were expressed in the wrong place. A mutation with that effect is a homeotic mutation, and homeotic is the word the homeobox is named after.

Apoptosis

Apoptosis is programmed cell death. Intracellular enzymes dismantle the cell in a controlled sequence while the cell-surface membrane remains intact. The cell fragments into membrane-bound apoptotic bodies, which are removed by phagocytosis without releasing their contents into the surrounding tissue.

Apoptosis occurs on a large scale in healthy tissue. Estimates for an adult human are of the order of ten thousand million cells a day, with mitosis replacing them.

The order of the steps is examinable. Enzymes inside the cell break down its cytoskeleton, so the cell loses its shape and shrinks, and the cytoplasm grows dense with organelles packed close together. The chromatin condenses, the nuclear envelope breaks down, and the DNA is cut into fragments. The cell surface membrane changes and begins to bleb, bulging outwards without tearing. The cell then breaks up into apoptotic bodies, each one a piece of the cell still wrapped in membrane. Finally those bodies are recognised and taken in by phagocytosis, and their contents are digested and the materials reused.

Everything in that sequence turns on the membrane staying whole. A cell killed by injury, by poison or by a shortage of oxygen swells instead, bursts, and spills hydrolytic enzymes and other contents into the tissue around it, which damages neighbouring cells and provokes inflammation. That is necrosis. State the contrast in those terms: apoptosis is controlled and does not release cell contents, whereas necrosis is uncontrolled and causes inflammation.

Apoptosis is switched on and off by cell signalling, from outside the cell and from inside it. Signals from outside include cytokines released by cells of the immune system, hormones, growth factors and nitric oxide, and the same signal can point either way depending on the cell that receives it: withdrawal of a growth factor initiates apoptosis as effectively as delivery of a cytokine does. Internal signals arise from the cell's own condition, and DNA damage that the repair systems cannot correct is the example to learn. Both routes converge on activation of the enzymes that carry out the sequence, so initiation is regulated while the sequence itself is stereotyped.

Apoptosis

Programmed cell death, in which a cell is broken down in an ordered sequence by its own enzymes and the fragments are removed by phagocytosis, without the release of the cell's contents.

Necrosis

Death of a cell caused by injury or by conditions it cannot survive, in which the cell swells, the membrane breaks and the contents escape and cause inflammation.

Apoptotic body

A membrane-bound fragment of a cell undergoing apoptosis, engulfed whole by a phagocyte.

Blebbing

The bulging of the cell surface membrane during apoptosis, which changes the shape of the cell without the membrane breaking.

Mitosis and apoptosis in digit development

Mitosis adds cells and apoptosis removes them, and a structure develops where the two are regulated relative to each other in space. Mitosis alone would produce a mass of tissue; apoptosis alone would remove it. The developing hand is the standard example of the two acting together.

In a human embryo the hand begins as a paddle. Cell division across the whole plate builds a flat, rounded lump of tissue with five thickenings inside it, and at that stage there are no separate fingers because the tissue between them is still there. Apoptosis then removes that tissue, cell by cell, along four strips running out from the wrist. No tissue is cut or reabsorbed by a neighbouring structure; the cells of the interdigital tissue receive a signal that initiates apoptosis, and phagocytes remove the fragments. What remains is five separate digits.

Mitosis forms the digit tissue; apoptosis removes cells between the developing digits. The digit count is the same before and after, because the digits were already specified by the regulatory genes described above; apoptosis removed the tissue connecting them.

The same pairing does the same job elsewhere. A tadpole's tail is removed by apoptosis during metamorphosis rather than being shed or digested from outside. A developing nervous system produces far more neurones than it keeps, and those that fail to make a working connection with a target cell no longer receive the growth factor that connection supplies, and die. In the immune system, lymphocytes that would react against the body's own tissue are removed by apoptosis before release. Next topic for that last example: the specific immune response.

Consequences of an imbalance between the two

A tissue maintains its size because the rate of mitosis and the rate of apoptosis are matched. A sustained difference between the two rates changes the tissue, and the two directions lead to different kinds of disease.

Consider too little apoptosis first. Prerequisite: the cell-cycle checkpoints. A cell held at a checkpoint with damage it cannot repair does not remain there indefinitely. It is normally removed by apoptosis, and the protein most often responsible is the product of a tumour suppressor gene called p53, which halts the cycle while repair is attempted and initiates apoptosis when repair fails. A cell therefore has two safeguards against passing on damaged DNA: it can stop dividing, and it can be removed. A mutation in the gene providing both removes both. The damaged cell divides, its daughters inherit the damage and the same defect, and further mutations accumulate in that lineage, which is one route to a tumour. Next topic: mutation, gene expression and cancer.

In the other direction, excessive apoptosis destroys tissue that is healthy and cannot be replaced. Neurones are the clearest case, because most of them have left the cell cycle for good, so a neurone lost is a neurone gone; excessive apoptosis of neurones is part of the damage in neurodegenerative disease and in the tissue around a stroke. The same logic explains why a drug that switched apoptosis off everywhere would be a poor idea however useful it sounded for protecting neurones, and why one that switched it on everywhere would be worse.

Two general points follow. Development is controlled by genes that regulate other genes, which is why relatively few are required and why one mutation can have such a large effect. And a body plan is produced by the removal of cells as well as by their proliferation, with the removal regulated as closely as the growth.